Naturally occurring human antibodies (immunoglobulin G (IgG), IgM, IgD, IgE, and IgA) are each present as an assembly of two heavy chains having the same amino acid sequence and two light chains having the same sequence. In this regard, homodimerization between the two identical heavy chains is induced by the non-covalent interactions between the constant region terminal domains (CH3 domains in IgG, IgD and IgA, CH4 domains in IgM, and CH2 and CH4 domains in IgE) and the disulfide bond between hinge domains.
Antibody heterodimeric Fc technology is a technology that makes heterodimeric fragment crystallizable (Fc) of immunoglobulin heavy chain constant regions by modifications to the CH3 domain interface, with different mutations on each domain such that the engineered Fc fragments, carrying the CH3 variant pair, preferentially form Fc heterodimers in naturally occurring antibodies (IgG, IgM, IgA, IgD, and IgE) rather than the Fc homodimers. More specifically, it is a technology that induces mutations in two different CH3 domains of Fc by genetic engineering, such that the two Fc fragments form a heterodimer with minimal sequence variations while they have tertiary structures very similar to those of naturally occurring antibodies (U.S. Pat. No. 7,695,936; and Korean Patent No. 1,522,954). The heterodimeric Fc technology is a platform technology for making bispecific antibodies, and CH3 domain mutants that induce Fc heterodimer formation known so far were mostly generated by introducing an asymmetric mutation pair into the CH3 domain interface by the structure-based rational design of antibody (Spreter Von Kreudenstein et al., 2014). Pioneering studies include knob-into-hole technology (Ridgway et al., 1996) from Genentech, and many multinational pharmaceutical companies, including Zymeworks (ZW1; Von Kreudenstein et al., 2013). Xencor (HA-TF; Moore G L et al., 2011) and EMD Serono (SEEDbody; Davis J H et al., 2010), have developed and reported the platform technology.
Above all, the A107 variant used in the present invention is a high-yield Fc heterodimer screened from a human antibody heterodimeric Fc library constructed using a yeast cell surface display system, and is a heterodimeric Fc variant which promotes the heterodimeric formation by inducing mutations at charged amino acids to form sterically complementary hydrophobic interactions (K409WCH3A-D399V/F405TCH3B) and forming hydrogen bonds (K370ECH3A-E357NCH3B), while retaining hydrophobic core integrity at the CH3 domain interface (Choi et al. 2016; Korean Patent Application No. 2015-0142181).
Heterodimeric Fc variants reported so far, including the A107 variant, are all based on IgG1 occupying the largest proportion of human antibody isotypes, and variants of isotypes (IgG2, IgG3, IgG4, IgA, IgM, and IgE) other than IgG1 have not been reported yet.
This is because therapeutic antibodies that are being marketed under approval of the U.S. Food and Drug Administration (FDA) mostly adopt the IgG1 isotype (Irani et al. 2015). In recent years, for immune-modulating antibodies or receptor agonist fusion proteins that do not need to have great antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cellular cytotoxicity (CDC), the development of therapeutic proteins based on IgG2 or IgG4 whose effector functions are significantly lower than those of IgG1 have been made.
Meanwhile, physiologically active proteins mostly have small sizes, and thus have the disadvantage of having a short in vivo half-life. In order to solve this disadvantage, there has been an attempt to conjugate PEG (polyethylene glycol) or the like, or fusion to an antibody Fc (crystallizable fragment) region. However, it has not yet been possible to develop physiologically active proteins whose activity is efficiently and sufficiently maintained for a long period of time.
In particular, for proteins composed of two or more different subunits, wherein the two or more different subunits form a protein complex to exhibit physiological activity, it has never been possible to develop Fc-fused proteins which are formed to have naturally occurring original protein complex structures with wild type Fc because wild type Fc-fused protein forms homodimer due to the homodimeric nature of Fc. Thus, wild type Fc is not suitable for Fc fusion for heterodimeric or heterooligomeric proteins to properly exhibit the activity of the original proteins and sufficiently maintain their activity for a long period of time.
Under this technical background, the present inventors have constructed heterodimer variants comprising Fc regions derived not only from IgG1, but also from other isotype antibodies such as IgG2, IgG3 and IgG4, which were previously not reported, and have used these heterodimer variants to develop a novel therapeutic fusion protein in the form of a heterodimeric Fc-fused protein wherein one or more subunits of a protein, which is composed of two or more different subunits and in which two or more subunits exhibit physiological activity by forming a protein complex, are genetically fused to the terminus of the Fc region, thereby completing the present invention.
In particular, in the present invention, preferably, interleukin-12 (IL-12) can be used as the protein which is composed of two different subunits, p35 and p40, wherein the two subunits exhibit physiological activity by forming the IL-12 protein.
IL-12 can directly kill tumors by increasing the activity of immune cells such as cytotoxic T lymphocytes (CTLs) or natural killer cells (NKs) among immune cells, or can inhibit tumorigenesis by activating immune responses through secretion of pro-inflammatory cytokines such as interferon-gamma (IFN-γ) in tumor microenvironments where the immune responses are inhibited. Thus, IL-12 has been much studied as an anti-cancer cytokine (Lasek et al., 2014). However, in the development of therapeutic methods using IL-12, the short half-life of the cytokine itself necessitates frequent administration which can lead to toxicity. For this reason, studies have been conducted to fuse IL-12 with an antibody or Fc in order to use it as long-acting IL-12 (Tugues et al., 2015). However, in these studies, a problem arises in that, due to the fusion of a wild-type Fc-based antibody that forms a homodimer by the interaction between CH3 domains, the fused IL12 protein is bivalent, unlike an endogenous monovalent form of IL-12, and for this reason, the wild type Fc-based antibody fused IL-12 shows poor physiological activity than endogenous IL-12, or unwanted localization appears due to avidity-driven increased binding of IL-12 to immune cells (Tzeng et al., 2015; Dumont et al., 2006).
Therefore, in an effort to make a monovalent fusion protein using a wild-type antibody or an Fc region, as shown in FIGS. 1(A) to 1(C), there has been used a method of constructing a fusion protein through a strategy such as fusing a selective tag for additional purification only to the C-terminus of a single Fc region or fusing an Fc region and a protein to each other after separately purifying them with high purity. However, this method is not only very costly to produce a large amount of protein, but also requires research to optimize an additional purification process.
However, the use of a heterodimeric Fc-fused protein according to the present invention makes it possible to easily produce a monovalent heterodimeric Fc-fused protein as shown in FIG. 2 without needing to optimize an additional purification process.